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An oligomer of the Sec61 trimeric complex is thought to form the protein-conducting channel for protein transport across the endoplasmic reticulum. A purified yeast Sec61 complex bound to monomeric yeast ribosomes as an oligomer in a saturable fashion. Cryo–electron microscopy of the ribosome-Sec61 complex and a three-dimensional reconstruction showed that the Sec61 oligomer is attached to the large ribosomal subunit by a single connection. Moreover, a funnel-shaped pore in the Sec61 oligomer aligned with the exit of a tunnel traversing the large ribosomal subunit, strongly suggesting that both structures function together in the translocation of proteins across the endoplasmic reticulum membrane.

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

A new technique for neutron scattering, the proton-spin contrast-variation, improves the signal-to-noise ratio more than one order of magnitude as compared to conventional techniques. The improved signal enables small RNA ligands within a large deuterated ribonucleic acid-protein complex to be measured. We used this technique to determine the positions of the two tRNAs within the elongating ribosome before and after translocation. Using a four-sphere model for each of the L-shaped tRNAs, unequivocal solutions were found for the localization of the mass centre of both tRNAs. The centre of gravity is located in the interface cavity separating the ribosomal subunits near the neck of the 30 S subunit. It moves during translocation by 12(±4) A towards the head of the 30 S subunit and slightly towards the L1 protuberance of the 50 S subunit.

Ribosomes translating secretory and membrane proteins are targeted to the endoplasmic reticulum membrane and attach to the protein-conducting channel and ribosome-associated membrane proteins (RAMPs). Recently, a new RAMP, ERj1p, has been identified that recruits BiP to ribosomes and regulates translational activity. Here we present the cryo-EM structure of a ribosome-ERj1p complex, revealing how ERj1p coordinates the ribosome at the membrane and how allosteric effects may mediate ERj1p's regulatory activity.

A method is proposed for determining the directions of projections. An arbitrary number of projections of unknown three-dimensional structure are simultaneously used as input. The method is based on common lines and uses a new discrepancy measure accounting for the uneven distribution of common lines in angular space. An application to the 70S Escherichia coli ribosome data obtained from an energy-filtering electron microscope is described.

Transfer RNA (tRNA) molecules play a crucial role in protein biosynthesis in all organisms. Their interactions with ribosomes mediate the translation of genetic messages into polypeptides. Three tRNAs bound to the Escherichia coli 70S ribosome were visualized directly with cryoelectron microscopy and three-dimensional reconstruction. The detailed arrangement of A- and P-site tRNAs inferred from this study allows localization of the sites for anticodon interaction and peptide bond formation on the ribosome.

An immunocomplex formed by the 4 × 6 meric hemocyanin of the scorpion Androctonus australis with the monoclonal antibody L104 was studied by cryoelectron microscopy and subjected to three-dimensional (3D) reconstruction. The reconstructed particle reflects the structure of the immunocomplex in its hydrated state and is devoid of the flattening that was previously observed with a negatively stained preparation. A 3D fitting of the X-ray data of the Panulirus interruptus hemocyanin and of the Fab R19.9 to the reconstruction volume allowed the first quantitative measurement of the structural parameters of the antigen, and the localization of the epitope at the surface of subunit Aa 6. The independent alignment of the Fobs and the hexamers provides a direct verification for the accuracy of the fit and allows the building of the most detailed model of a cheliceratan 4 × 6meric complex and its attached monoclonal antibodies.

A computer program for automatic particle picking based on textural methods is proposed. The technique relies on the evaluation of certain textural parameters for data windows containing single particles, and those containing undesirable material. These parameters are manipulated by a discriminant analysis routine for determining the rules of classification between the different categories. The effectiveness of the method was demonstrated by application to electron micrographs of 70S Escherichia coli ribosomes.

The three-dimensional organization of the internal compartments of conventionally fixed and embedded rat-liver mitochondria has been determined by tomographic reconstruction from tilt-series images collected on the Albany high-voltage electron microscope. The results indicate that the inner membranes of these organelles are predominantly tubular in the orthodox (expanded matrix) conformation, as previously suggested by scanning electron microscopy. In the condensed (contracted matrix) conformation, the intracristal space opens up into large irregularly shaped compartments which are connected to each other and to the external (intermembrane) space by tubes with approximately the same diameter (20 nm) as those observed in the orthodox state. These results raise several questions, in particular about the nature of the structural transitions that occur in the cristae during matrix expansion and contraction, and about the influence of inner-membrane shape on the diffusion of ions and metabolites between the intracristal and intermembrane compartments. © 1994 Wiley-Liss, Inc.

Cryo-electron microscopy of single biological particles opens up new possibilities for structure analysis: the particle can be reconstructed in its native shape and internal features are preserved. To take advantage of these possibilities we have developed new methods of data collection and image processing and we have applied them to the 70S Escherichia coli ribosome. A method of orientation search is proposed, which makes it possible to relate random-conical data sets to one another even if they are collected from low-tilt micrographs. A technique for 3D alignment of projections is described and applied to the single-micrograph 3D reconstruction.

A frozen-hydrated sample embedded in vitreous ice of human α2-macroglobulin transformed by methylamine was imaged by cryoelectron microscopy and reconstructed in three dimensions. In the reconstruction, the cage-like architecture of this protease inhibitor is fully revealed with a clear visualization of two lozenge-shaped lateral walls connected by thin bridges. The shape and dimensions of the internal cavity normally containing the trapped protease(s) is described. The possible locations of the thiol ester sites and intersubunit connections are also discussed.

Three-dimensional cryo-electron microscopy (EM) of macromolecular assemblies with low or nonexistent symmetry in single-particle form is now recognized as a powerful, legitimate method of structure research. The progress in the resolution achieved has always been related to the introduction of new image processing methods and to improvements in understanding of the overall strategy of single particle analysis. The new techniques either were adopted from methods developed in other fields, or were specifically designed to deal with extremely low Signal-to-Noise Ratio (SNR) data in EM. In the former category we can place multivariate statistical analysis, clustering techniques, correlation techniques, and tomographic methods, in the latter alignment methods, three-dimensional (3D) projection alignment, contrast transfer function (CTF) correction methods, and common-lines based orientational search. In addition, there have been steady improvements of the quality of the data by seeking better electron microscopy conditions (usage of higher microscope voltage and of microscopes equipped with a field emission gun).

We describe a fast and accurate direct Fourier method for reconstructing a function f of three variables from a number of its parallel beam projections. The main application of our method is in single particle analysis, where the goal is to reconstruct the mass density of a biological macromolecule. Typically, the number of projections is extremely large, and each projection is extremely noisy. The projection directions are random and initially unknown. However, it is possible to determine both the directions and f by an iterative procedure; during each stage of the iteration, one has to solve a reconstruction problem of the type considered here. Our reconstruction algorithm is distinguished from other direct Fourier methods by the use of gridding techniques that provide an efficient means to compute a uniformly sampled version of a function g from a nonuniformly sampled version of Fg, the Fourier transform of g, or vice versa. We apply the two-dimensional reverse gridding method to each available projection of f, the function to be reconstructed, in order to obtain Ff on a special spherical grid. Then we use the three-dimensional gridding method to reconstruct f from this sampled version of Ff. This stage requires a proper weighting of the samples of Ff to compensate for their nonuniform distribution. We use a fast method for computing appropriate weights that exploits the special properties of the spherical sampling grid for Ff and involves the computation of a Voronoi diagram on the unit sphere. We demonstrate the excellent speed and accuracy of our method by using simulated data.

The present-day interactive manual selection of biological molecules from digitized micrographs for single particle averaging and reconstruction requires substantial effort and time. Thus a computer algorithm capable of recognition of structural content and selection of particles would be desirable.A few approaches have been proposed in the past. The method by Frank and Wagenknecht is based on the principle of correlation search. Van Heel's method is based on the computation of the local variance over a small area around each point of the image field. The method by Harauz and Fong-Lochovsky is based on edge-detection. The present work was focussed on the detection and classification of particlesby exploiting the standard statistical methods of discriminant analysis.The proposed technique is described in the block diagram (Fig.1). As illustrated in the figure, the program consists of three distinct segments devoted to, respectively, preparation of the data, training session and automatic selection based on a discriminant function set up in the training, hi the data preparation segment, the micrograph is (i) reduced four-fold in size, (ii) low-pass filtered and (iii) run through a peak search algorithm.

We have obtained two reconstructions of the 70S ribosomal subunit embedded in ice using the random-conical method on particles showing two distinctive views.Ribosomes were applied to grids covered with thick holey carbon film whose holes were in turn covered with a second, thin carbon film. The frozen-hydrated preparation was done as described previously using GATAN cryo-transfer equipment and specimen holder on a Philips EM420. The images were taken in pairs at 2μm defocus and a dose of less than 10 e-1/Å2. The first was taken with the specimen tilted to 50°, the second untilted. A total of 838 particles from two micrograph pairs were selected for image analysis.A new reference-free alignment algorithm was used to align the 0°-images. In the subsequent multivariate statistical analysis and factor-space based hierarchical classification, several distinct groups were found, two of which (averages, Fig. 1) with memberships above 10% were selected for two separate 3D reconstructions. The averages were found to have a resolution of 3.3 nm as established by phase residual analysis. For computing the reconstruction, we used an iterative scheme that is based on a Chi-square minimization. In this way, we were able to reduce distortions caused by the missing cone as well as by large gaps in angular spacing.

The aims and merits of the syntactical decomposition of the epileptic EEG are discussed in some detail. General assumptions in the design of segmenting algorithm - an essential part of the reduction of data program - are considered and a working example of segmenting routine os presented. This routine is designed as a finite state automaton, equivalent to a formal regular grammar. The performance of the routine on two sets of experimental data is presented and its intrinsic stability is shown in terms of cluster analysis of the segments produced. Means of extension of the proposed scheme to other cases of potential interest as well as possible applications in the higher level syntactical analysis are pointed out.

This paper discusses in detail some properties of Sugeno's g λ measure. A clustering algorithm making use of these properties is presented and its performance, when run on the well-known set of the iris data, is briefly described. The inherent advantages of the approach proposed are pointed out in the concluding part of the paper.

A full-scale scheme of the multi-level syntactic analysis of the epileptic EEG is presented and discussed in detail. An exhaustive description of basic procedures necessary to obtain a meaningful description of any set of epileptic EEG recordings is given; these procedures include algorithms for extraction of various elementary peaks from the EEG signal, for segmentation of the recording, for classification of the segments found into distinct groups of characteristic patterns and, finally, for calculation of the Levenshtein distance between the individual recordings, each recording regarded as a string of symbols taken to represent different classes of patterns. An example of global analysis in terms of such a distance, performed on a typical set of epileptic EEG recordings, is supplied to prove the feasibility of the scheme proposed.

Fragmentation of molecules and their representation based on structural pattern is suggested in order to predict the retention characteristics in HPLC. The logarithm of the capacity factor is derived as a sum of logarithms of the structural fragments. The retention behaviour of a polycyclic aromatic hydrocarbon is related to the numerical representation of the individual compounds. In all cases a high correlation coefficient, ranging from 0.97 to 0.99 was obtained. The contribution of the molecule fragments to the capacity factor value is discussed.

A multi-level scheme of syntactic reduction of the epileptiform EEG data is briefly discussed and the possibilities it opens up in describing the dynamic behaviour of a multi-channel system are indicated. A new algorithm for the inference of a Markov network from finite sets of sample symbol strings is introduced. Formulae for the time-dependent state occupation probabilities, as well as joint probability functions for pairs of channels, are given. An exemplary case of analysis in these terms, taken from an investigation of anticonvulsant drug effects on EEG seizure patterns, is presented.

Proteasome-dependent proteolysis is essential for a number of key cellular processes and requires a sophisticated biogenesis pathway to function. Here, we have arrested the assembly process in its dynamic progression at the short-lived 16S state. Structural analysis of the 16S proteasome precursor intermediates by electron microscopy, and single particle analysis reveals major conformational changes in the structure of the β-ring in comparison with one-half of the 20S proteasome. The individual β-subunits in the 16S precursor complex rotate with respect to their positions in the x-ray crystallographic structure of the fully assembled 20S. This rearrangement results in a movement of the catalytic residue threonine-1 from the protected location in 16S precursor complexes to a more exposed position in the 20S structure. Thereby, our findings provide a molecular explanation for the structural rearrangements necessary for the dimerization of two 16S precursor complexes and the subsequent final maturation to active 20S proteasomes.

Macromolecular structure determination by cryo-electron microscopy (EM) and single particle analysis are based on the assumption that imaged molecules have identical structure. With the increased size of processed datasets it becomes apparent that many complexes coexist in a mixture of conformational states or contain flexible regions. As the cryo-EM data is collected in form of projections of imaged molecules, the information about variability of reconstructed density maps is not directly available. To address this problem, we describe a new implementation of the bootstrap resampling technique that yields estimates of voxel-by-voxel variance of a structure reconstructed from the set of its projections. We introduced a novel highly efficient reconstruction algorithm that is based on direct Fourier inversion and which incorporates correction for the transfer function of the microscope, thus extending the resolution limits of variance estimation. We also describe a validation method to determine the number of resampled volumes required to achieve stable estimate of the variance. The proposed bootstrap method was applied to a dataset of 70S ribosome complexed with tRNA and the elongation factor G. The variance map revealed regions of high variability: the L1 protein, the EF-G and the 30S head and the ratchet-like subunit rearrangement. The proposed method of variance estimation opens new possibilities for single particle analysis, by extending applicability of the technique to heterogeneous datasets of macromolecules, and to complexes with significant conformational variability.

Summary Identification of homogeneous subsets of images in a macromolecular electron microscopy (EM) image data set is a critical step in single-particle analysis. The task is handled by iterative algorithms, whose performance is compromised by the compounded limitations of image alignment and K-means clustering. Here we describe an approach, iterative stable alignment and clustering (ISAC) that, relying on a new clustering method and on the concepts of stability and reproducibility, can extract validated, homogeneous subsets of images. ISAC requires only a small number of simple parameters and, with minimal human intervention, can eliminate bias from two-dimensional image clustering and maximize the quality of group averages that can be used for ab initio three-dimensional structural determination and analysis of macromolecular conformational variability. Repeated testing of the stability and reproducibility of a solution within ISAC eliminates heterogeneous or incorrect classes and introduces critical validation to the process of EM image clustering.

An extension of the Kalman filter algorithm to the multi-channel case is presented and its application as a segmenting procedure in the analysis of the epileptic EEG is discussed. An analytical example of structural analysis, using the segments extracted by the proposed filter, is presented for a particular set of 4-channel EEG recordings. This analysis is shown to be especially fruitful if the autoregressive coefficients - a by product of the filtering procedure - are used to estimate the information flow between the channels by the calculation of partial as well as directed coherences for the representative segments.

A new algorithm for image reconstruction is proposed. Because a varying sampling increment is introduced, the interpolation during reconstruction is eliminated and the Fourier transform from modified polar to Cartesian sample grid is applied. The comparison of the back-projection of filtered projections method with the proposed PCFT algorithm is performed on computer-simulated data. Good resolution of the reconstructed images and a relatively short reconstruction time are the merits of the newly developed technique NMR imaging is cited as a possible field of application of the algorithm.